Characterization by high-performance liquid chromatography of circulating growth hormone-releasing factors in normal...

Four forms of circulating immunoreactive human GH-releasing factor (ir-hGRF) have been identified in each of four normal subjects, with a mean increase in total ir-hGRF of twofold over basal levels following a mixed meal. Plasma samples (200 ml) from each individual were subjected to large-scale Vycor extraction with initial purification by high-performance liquid chromatography on a reversed-phase C18 column, followed by analytical separation of the ir-hGRF components using a C3 wide-pore reversed-phase column, and subsequent radioimmunoassay of the fractions. The mean recovery of total ir-hGRF from the plasma (fasted and non-fasted) was 76 +/- 16% (2 X S.E.M.). Analytical separation of the ir-hGRF revealed four components which co-eluted with synthetic hGRF-37, hGRF-40 and hGRF-44, and a peak eluting between hGRF-40 and -44 which may represent hGRF-42. The hGRF-40 was shown to be the predominant circulating molecular form in the fasted state in each subject, and in three out of four subjects following a mixed meal. The hGRF-44 showed the greatest percentage increase over basal in all four individuals.